RNA amidates and thioamidates for RNAi

ABSTRACT

The present disclosure relates to RNA amidates and thioamidates useful for RNA interference applications. The RNA amidates and thioamidates contain at least one internucleoside linkage chosen from ribo-N3′→P5′ phosphoramidate (NP) and ribo-N3′→P5′ thiophosphoramidate (NPS) linkages, and optionally further containing at least one covalently conjugated lipid moiety. Compositions comprising the amidates and thioamidates are disclosed, as are methods for their use in modulating gene expression.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/578,530, now issued as U.S. Pat. No. 9,133,233 on Sep. 15, 2015,which is a 371 of International Application No. PCT/US04/032780 filedNov. 3, 2004, which claims the priority benefit of U.S. ProvisionalApplication No. 60/516,769 filed Nov. 4, 2003.

DESCRIPTION OF THE INVENTION

Field of the Invention

The present invention relates to the use of N3′→P5′ phosphoramidate (NP)and N3′→P5′ thiophosphoramidate (NPS) oligonucleotide chemistry for RNAinterference, optionally including the addition of covalently linkedlipid groups. N3′→P5′ NP and N3′→P5′ NPS chemistry confers superiorstability characteristics on the molecules, and the optional addition oflipid groups confers superior cellular uptake.

Background of the Invention

Several kinds of potential nucleic acid therapeutics have been exploredover the last two decades, including RNA inhibitors such as antisense,ribozymes (catalytic RNAs), and artificial ligand inhibitors(“aptamers”). These therapeutics are designed to silence geneexpression, and thus to alleviate the effects of undesirable genes, bethey endogenous to an organism or exogenous, such as bacterial or viralin origin. Because it is difficult to apply these to cells externally,there has been significant interest in expressing them within cells.However, expression of these therapeutics intracellularly has provedquite difficult as well; this difficulty is thought to be due to severalfactors. These include, for RNA-based therapeutics as an example, theconsiderations of finding their targets, folding into the effectiveconfiguration, and possibly interacting with the appropriate proteinswhile avoiding interactions with inappropriate proteins. There have beenisolated promising results (see, for example, Bertrand, E. et al.,RNAS3: 75-88 (1997); Good, P D et al. Gene Therapy 4:45-54 (1997)), butno therapeutics have yet resulted.

RNA Interference

RNA interference, or RNAi, is an endogenous, efficient, and potentgene-specific silencing technique that uses double-stranded RNAs (dsRNA)to mark a particular transcript for degradation in vivo. Firstdiscovered in the nematode Caenorhabditis elegans, it has since beenfound to operate in a wide variety of organisms. RNAi is believed to beeffected by dsRNAs ˜21-25 nucleotides long, called short interferingRNAs (siRNAs), which are endogenously produced through the degradationof long dsRNA molecules by an RNAse III-related nuclease called Dicer.Once formed, the siRNAs associate with a multiprotein complex calledRISC (RNA-Induced Silencing Complex), which targets the homologous RNAby Watson-Crick base pairing for sequence specific degradation of mRNA.

This sequence-specific degradation of mRNA results in knocking down(partially or completely) the targeted gene. Thus RNAi provides analternative to presently available methods of knocking down (or out) agene or genes. This method of knocking down gene expression can be usedtherapeutically or for research (e.g., to generate models of diseasestates, to examine the function of a gene, to assess whether an agentacts on a gene, or to validate targets for drug discovery).

There are two main approaches to employing RNAi in cells. In the firstapproach, an expression construct (for either integrative or transientexpression), which encodes an RNA including the desired RNAi sequences,is introduced into the target cells. The endogenous dicer enzymerecognizes and processes this RNA into the desired ˜21-23 nucleotidesiRNAs, which then enter an effector complex, RISC. In the secondapproach, the siRNAs (in either single-stranded antisense ordouble-stranded form) are introduced directly into the cell and directlyenter the RISC complex. In both of these approaches, guided by theantisense strand of the siRNA, the active form of RISC (activated by theATP-dependent unwinding of the siRNA duplex) recognizes and suppressesgene expression through mRNA degradation or prevention of proteinsynthesis.

RNAi has been studied in a variety of systems. Fire et al., Nature, 391:806 (1998), were the first to observe RNAi in C. elegans. Wianny andGoetz, Nature Cell Biol., 2:70 (1999), describe RNAi mediated by dsRNAin mouse embryos. Hammond et al., Nature, 404:293 (2000), describe RNAiin Drosophilia cells transfected with dsRNA. Elbashir at al., Nature,411:494 (2001), describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells by includinghuman duplexes of synthetic 21-nucleotide RNAs in cultured mammaliancells including embryonic kidney and HeLa cells.

Recent work in Drosophilia embryonic lysates (Elbashir et al., EMBO J.,20:6877(2001)) has revealed certain requirements for siRNA length,structure, chemical composition, and sequence that are essential tomediate efficient RNAi activity. These studies have shown that21-nucleotide siRNA duplexes are most active when containing 3′-terminaldinucleotide overhangs. Furthermore, complete disubstitution of one orboth siRNA strands with 2′deoxy (2′-H) or 2′-O-methyl nucleotidesabolishes RNAi activity, whereas substitution of the 3′-terminal siRNAoverhang nucleotides with 2′deoxy nucleotides (2′-H) was shown to betolerated. Single mismatch sequences in the center of the siRNA duplexwere also shown to abolish RNAi activity. In addition, these studiesalso indicate that the position of the cleavage site in the target RNAis defined by the 5′-end of the siRNA guide sequence rather than the3′-end of the guide sequence (Elbashir et al., EMBO J., 20:6877 (2001)).Other studies have indicated that a 5′-phosphate on the targetcomplementary strand of a siRNA duplex is required for siRNA activityand that ATP is utilized to maintain the 5′-phosphate moiety on thesiRNA (Nykanen et al., Cell, 107:309 (2001)).

Studies have shown that replacing the 3′-terminal nucleotide overhangingsegments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangswith deoxyribonucleotides does not have an adverse effect on RNAiactivity. Replacing up to 4 nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well-tolerated, whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity. In addition, Elbashir et al., supra, also report thatsubstitution of siRNA with 2′-O-methyl nucleotides completely abolishesRNAi activity. Li et al., International PCT Publication No. WO 00/44914,and Beach et al., International PCT Application No. WO 01/68836preliminarily suggest that siRNA may include modifications to either thephosphate sugar backbone or the nucleoside to include at least one of anitrogen or sulfur heteroatom; however, neither application postulatesto what extent such modifications would be tolerated in siRNA molecules,nor provides any further guidance or examples of such modified siRNA.Kreutzer et al., Canadian Patent Application No. 2,359,180, alsodescribe certain chemical modifications for use in dsRNA constructs inorder to counteract activation of double stranded RNA-dependent proteinkinase PKR, specifically 2′-amino or 2′-O-methyl nucleotides, andnucleotides containing a 2′-O or 4′-C methylene bridge. However, it isunclear as to what extent these modifications would be tolerated insiRNA molecules.

The use of longer dsRNA has been described. For example, Beach et al.,International PCT Publication No. WO 01/68836, describes specificmethods for attenuating gene expression using endogenously-deriveddsRNA. Tuschl et. al. International PCT Publication No. WO 01/75164,describe a Drosophilia in vitro RNAi system and the use of specificsiRNA molecules for certain functional genomic and certain therapeuticapplications; although Tuschl, Chem. Biochem., 2:239-245 (2001), doubtsthat RNA can be used to cure genetic diseases or viral infection due tothe danger of activating interferon response. Li et al., InternationalPCT Publication No. 01/36646, describe certain methods for inhibitingthe expression of particular genes in mammalian cells using dsRNAmolecules. Fire et al., International PCT Publication No. WO 99/32619,describe particular methods for introducing certain dsRNA molecules intocells for use in inhibiting gene expression. Plaetinck et al.,International PCT Publication No. WO 00/01846, describe certain methodsfor identifying specific genes responsible for conferring a particularphenotype in a cell using specific dsRNA molecules. Mello et al.,International PCT Publication No. WO 01/29058, describe theidentification of specific genes involved in dsRNA-mediated RNAi.Deschamps Depaillette et al., International PCT Publication No. WO99/07409, describe specific compositions consisting of particular dsRNAmolecules combined with certain anti-viral agents. Waterhouse et al.,International PCT Publication No. 99/53050, describe certain methods fordecreasing the phenotypic expression of a nucleic acid in plant cellsusing certain dsRNAs. Driscoll et al., International PCT Publication No.WO 01/49844, describe specific DNA constructs for use in facilitatinggene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. Forexample, Parrish et al., Molecular Cell, 6:1977-1087 (2000), describechemically modified siRNA constructs targeting the unc-22 gene of C.elegans. Grossniklaus, International PCT Publication No. WO 01/38551,describes certain methods for regulating polycomb gene expression inplants using certain dsRNAs. Churikov et al., International PCTPublication No. WO 01/38551, describe certain methods for modifyinggenetic characteristics of an organism using certain dsRNAs. Cogoni etal., International PCT Publication No. WO 01/53745, describe certainmethods for isolating a neurospora silencing gene and uses thereof. Reedet al., International PCT Publication No. WO 01/70944, describe certainmethods of drug screening using transgenic nematodes as Parkinson'sDisease models using certain dsRNAs. Deak et al., International PCTPublication No. WO 01/72774, describe certain Drosophilia-derived geneproducts that may be related to RNAi in Drosophilia. Arndt et al.,International PCT Publication No. WO 01/92513 describe certain methodsfor mediating gene suppression by using factors that enhance RNAi.Tuschl et al., International PCT Publication No. WO 02/44321, describecertain synthetic siRNA constructs. Pachuk et al., International PCTPublication No. WO 00/6334, and Satishchandran et al., International PCTPublication No. WO 01/04313, describe certain methods and compositionsfor inhibiting the function of certain polynucleotide sequences usingcertain dsRNAs. Echeverri et al., International PCT Publication No. WO02.38805, describe C. elegans genes identified via RNAi. Kruetzer etal., International PCT Publications Nos. WO 02/055692, WO 02/055693, andEP 1144623 B1 describe certain methods for inhibiting gene expressionusing RNAi. Graham et al., International PCT Publication Nos. WO99/49029 and WO 01/70949, and AU 4037501 describe certain vectorexpressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559,describe certain methods for inhibiting gene expression in vitro usingcertain long dsRNA (greater than 25 nucleotide) constructs that mediateRNAi.

Delivering siRNAs directly to whole vertebrate animals is moreproblematic than it is for invertebrates or cell lines. Conventionallyconstructed oligonucleotides have poor serum stability, are susceptibleto nuclease degradation, and cannot easily cross cell membranes. Twogroups of scientists independently employed a “hydrodynamic transfectionmethod” to deliver naked siRNAs to mice via tail-vein injection. A. P.McCaffrey et al., “Gene expression: RNA interference in adult mice,”Nature, 418:38-9 (2002); D. J. Lewis et al., “Efficient delivery ofsiRNA for inhibition of gene expression in postnatal mice,” Nat. Genet.,32:107-8 (2002). While these scientists observed downregulation of areporter gene by 80%-90% in the liver, kidney, spleen, lung, andpancreas, the effect was relatively short-lived, lasting only a fewdays.

Thus, there is a need to produce siRNAs that have improvedcharacteristics for both in vitro delivery to cells and in particular,in vivo delivery for therapeutic applications.

The design of nucleic acids, particularly oligonucleotides, for in vivodelivery requires consideration of various factors including bindingstrength, target specificity, serum stability, resistance to nucleasesand cellular uptake. A number of approaches have been proposed in orderto produce oligonucleotides that have characteristics suitable for invivo use, such as modified backbone chemistry, formulation in deliveryvehicles and conjugation to various other moieties. Therapeuticoligonucleotides with characteristics suitable for systemic deliverywould be particularly beneficial.

Oligonucleotides with modified chemical backbones are reviewed inMicklefield, Backbone modification of nucleic acids: synthesis,structure and therapeutic applications, Curr. Med. Chem., 8(10):1157-79,2001 and Lyer et al., Modified oligonucleotides-synthesis, propertiesand applications, Curr. Opin. Mol. Ther., 1(3): 344-358, 1999.

Examples of modified backbone chemistries include:

-   peptide nucleic acids (PNAs) (see Nielsen, Methods Mol. Biol.,    208:3-26, 2002),-   locked nucleic acids (LNAs) (see Petersen & Wengel, Trends    Biotechnol., 21(2):74-81, 2003),-   phosphorothioates (see Eckstein, Antisense Nucleic Acid Drug Dev.,    10(2):117-21, 2000),-   methylphosphonates (see Thiviyanathan et al., Biochemistry,    41(3):827-38, 2002),-   phosphoramidates (see Gryaznov, Biochem. Biophys. Acta,    1489(1):131-40, 1999; Pruzan et al., Nucleic Acids Res.,    30(2):559-68, 2002), and-   thiophosphoramidates (see Gryaznov et al., Nucleosides Nucleotides    Nucleic Acids, 20(4-7):401-10, 2001; Herbert et al., Oncogene, 21    (4):638-42, 2002).

Each of these types of oligonucleotides has reported advantages anddisadvantages. For example, peptide nucleic acids (PNAs) display goodnuclease resistance and binding strength, but have reduced cellularuptake in test cultures; phosphorothioates display good nucleaseresistance and solubility, but are typically synthesized as P-chiralmixtures and display several sequence-non-specific biological effects;methylphosphonates display good nuclease resistance and cellular uptake,but are also typically synthesized as P-chiral mixtures and have reducedduplex stability. The N3′→P5′ phosphoramidate internucleoside linkagesare reported to display favorable binding properties, nucleaseresistance, and solubility (Gryaznov and Letsinger, Nucleic AcidsResearch, 20:3403-3409, 1992; Chen et al., Nucleic Acids Research,23:2661-2668, 1995; Gryaznov et al., Proc. Natl. Acad. Sci.,92:5798-5802, 1995; Skorski et al., Proc. Natl. Aced. Sci.,94:3966-3971, 1997). However, they also show increased acid labilityrelative to the natural phosphodiester counterparts (Gryaznov et al.,Nucleic Acids Research, 24:1508-1514, 1996). Acid stability of anoligonucleotide is an important quality given the desire to useoligonucleotide agents as oral therapeutics. The addition of a sulfuratom to the backbone in N3′→P5′ thiophosphoramidate oligonucleotidesprovides enhanced acid stability.

As with many other therapeutic compounds, the polyanionic nature ofoligonucleotides reduces the ability of the compound to cross lipidmembranes, limiting the efficiency of cellular uptake. Various solutionshave been proposed for increasing the cellular uptake of therapeuticagents, including formulation in liposomes (for reviews, see Pedroso deLima et al., Curr. Med. Chem., 10(14):1221-1231, 2003 and Miller, Curr.Med. Chem., 10(14):1195-211, 2003) and conjugation with a lipophilicmoiety. Examples of the latter approach include: U.S. Pat. No. 5,411,947(Method of converting a drug to an orally available form by covalentlybonding a lipid to the drug); U.S. Pat. No. 6,448,392 (Lipid derivativesof antiviral nucleosides: liposomal incorporation and method of use);U.S. Pat. No. 5,420,330 (Lipo-phosphoramidites); U.S. Pat. No. 5,763,208(Oligonucleotides and their analogs capable of passive cell membranepermeation); Gryaznov & Lloyd, Nucleic Acids Research, 21:5909-5915,1993 (Cholesterol-conjugated oligonucleotides); U.S. Pat. No. 5,416,203(Steroid modified oligonucleotides); WO 90/10448 (Covalent conjugates oflipid and oligonucleotide); Gerster et al., Analytical Biochemistry,262:177-184 (1998) (Quantitative analysis of modified antisenseoligonucleotides in biological fluids using cationic nanoparticles forsolid-phase extraction); Bennett et al., Mol. Pharmacol., 41:1023-1033(1992) (Cationic lipids enhance cellular uptake and activity ofphophorothioate antisense oligonucleotides); Manoharan et al., Antisenseand Nucleic Acid Drug Dev., 12:103-128 (2002) (Oligonucleotideconjugates as potential antisense drugs with improved uptake,biodistribution, targeted delivery and mechanism of action); and Fiedleret al., Langenbeck's Arch. Surg., 383:269-275 (1998) (Growth inhibitionof pancreatic tumor cells by modified antisense oligodeoxynucleotides).

SUMMARY OF THE INVENTION

This invention aids in fulfilling these needs in the art. In accordancewith the invention, there is provided a small interfering RNA comprising15-25 nucleotides complementary to a target nucleic acid sequence,wherein the RNA comprises at least one internucleoside linkage chosenfrom ribo-N3′→P5′ phosphoramidate (NP) and ribo-N3′→P5′thiophosphoramidate (NPS) linkages.

According to another aspect of the invention, there is provided acompound comprising the structure O-(x-L)_(n), wherein O is ariboamidate of formula:

R₁ is chosen from fluorine and OR₂, R₂ is chosen from hydrogen and loweralkyl, B is chosen from purines, pyrimidines, and analogs thereof, and Zis chosen from oxygen and sulfur, and further wherein the riboamidatecomprises a sequence of 15 to 25 bases, and said sequence is at leastpartially complementary to a selected target sequence; L is a lipidmoiety; x is an optional linker; and n is an integer ranging from 1 to5, wherein if n>1, each additional (x-L) component may be,independently, the same or different.

According to still another aspect of the invention, there is provided amethod for effecting the post-transcriptional silencing of at least onegene, comprising administering to a mammal in need of suchpost-transcriptional silencing at least one a small interfering RNAcomprising 15-25 nucleotides complementary to a target nucleic acidsequence, wherein the RNA comprises at least one internucleoside linkagechosen from ribo-N3′→P5′ phosphoramidate (NP) and ribo-N3′→P5′thiophosphoramidate (NPS) linkages.

According to yet another aspect of the invention, there is provided amethod for regulating the expression of genes in an organism, comprisingadministering to a mammal in need of such regulation at least one smallinterfering RNA comprising 15-25 nucleotides complementary to a targetnucleic acid sequence, wherein the RNA comprises at least oneinternucleoside linkage chosen from ribo-N3′→P5′ phosphoramidate (NP)and ribo-N3′→P5′ thiophosphoramidate (NPS) linkages.

According to still a further aspect of the present invention, there isprovided a single-stranded small interfering RNA that inhibits theexpression of an endogenous mammalian target RNA sequence, wherein thesingle-stranded small interfering RNA comprises at least oneinternucleoside linkage chosen from ribo-N3′→P5′ phosphoramidate (NP)and ribo-N3′→P5′ thiophosphoramidate (NPS) linkages.

The compositions and methods of the present invention relate to RNAamidates and thioamidates, optionally comprising at least one covalentlylinked lipid group, for RNAi applications. The compounds of theinvention have superior cellular uptake properties. This means anequivalent biological effect can be obtained using smaller amounts ofoligonucleotide. When applied to the human therapeutic setting, this cantranslate to reduced toxicity risks and cost savings. The compounds ofthe invention knock-out gene expression by RNA interference, e.g., bymediating interference of mRNA.

The mRNA of any gene can be targeted for degradation using the methodsof mediating interference of mRNA. For example, any cellular or viralmRNA can be targeted and, as a result, the encoded protein (e.g., anoncoprotein, a viral protein), expression will be diminished. Inaddition, the mRNA of any protein associated with, or causative of, adisease or undesirable condition can be targeted for degradation.

For example, the compounds disclosed herein can be designed and used tomodulate or block: Hepatitis B virus (HBV) and Hepatitis C virus (HCV)protein expression, and can thus be used to treat diseases associatedwith HBV and HCV such as, for example, cirrhosis, liver failure, andhepatocellular carcinoma; Ras gene expression, such as K-Ras (associatedwith colon and pancreatic carcinomas), H-Ras (associated withleukemias), and/or N-Ras expression; HIV-1 and HER2 gene expression, thelatter of which is associated with breast and ovarian cancers;expression of vascular endothelial growth factor and/or vascularendothelial growth factor receptors, such as VEGFR1 and/or VEGFR2, forthe purpose of, e.g., preventing, treating, controlling disorders andconditions related to angiogenesis, including but not limited to cancer,tumor angiogenesis, or ocular indications, such as diabetic retinopathy,or age-related macular degeneration, proliferative diabetic retinopathy,hypoxia-induced angiogenisis, rheumatoid arthritis, psoriasis, woundhealing, endometriosis, endometrial carcinoma, gynecologic bleedingdisorders, irregular menstrual cycles, ovulation, premenstrual syndrome(PMS), and menopausal dysfunction; beta-secretase (BACE), PIN-1,presenillin-1 (PS-1) and presenillin-2 (PS-2) polypeptide andpolynucleotide targets, associated with Alzheimer's disease; expressionof NOGO and NOGO receptor genes, and the expression of genes encodingthe IκB kinase IKK complex, for example IKK-alpha, IKK-beta, orIKK-gamma and/or a protein kinase PKR protein; expression of kinaseswhich phosphorylate Cdc25 S216, such as Chk1 (checkpoint kinase 1)enzyme, Chk2 (Cds1) and C-Tak1; and expression of the T-cellco-stimulatory adapter protein GRID (Grb2-related with Insert Domain).

In an exemplary therapeutic application, a 19 to 23 nucleotideriboamidate, such as a 21 to 23 nucleotide riboamidate, is introducedinto a mammal or mammalian cells, for example a human or human cells, inorder to mediate RNA interference in the mammal or mammalian cells, suchas to prevent or treat a disease or undesirable condition. In thismethod, a gene (or genes) that cause or contribute to the disease orundesirable condition is targeted and a riboamidate complementary to themRNA of the gene targeted for degradation is introduced into the cell ororganism. The cell or organism is maintained under conditions underwhich degradation of the corresponding mRNA occurs, thereby mediatingRNA interference of the mRNA in the gene in the cell or organism.

Two forms of RNAi are provided, a single-stranded form and adouble-stranded form. Single-stranded forms are antisense (complementaryto the coding strand of the targeted message) and are typically at least17 bases in length, up to 50 bases in length, more usually from about 19to about 25 bases in length, for example, from 19 to 23 bases in length.These single-stranded forms are suitably constituted with 100%riboamidates (NP or NPS), but can include other linkage forms, such asphosphodiester, and can also include some DNA nucleobases such as, forexample, uracil, thymine, adenine, guanine, cytosine, and analogsthereof. The optional linkage of one or more lipid moieties is suitablyto the 3′ amino terminus or 5′ terminus, but can also be to anucleobase.

Double-stranded forms contain the sense and antisense regions and havethe same size constraints as the single-stranded forms. They can beblunt-ended, or can include a 3′ overhang to increase resistance toendonucleases. 5′ overhangs are also possible. The antisense strand isthe effector moiety, and is suitably entirely composed of riboamidates,but can also include some other linkage forms, including DNAnucleobases. The sense strand is less critical and other chemistries,including DNA, can be suitably employed.

BRIEF DESCRIPTION OF THE DRAWINGS

This invention will be described with reference to the drawings inwhich:

FIG. 1, comprising chemical structures 1A to 1DD, shows examples of theattachment of various lipid groups (L) to oligonucleotides in compoundsof the invention. In FIG. 1, R₁ is chosen from fluorine and OR₂, whereinR₂ is chosen from hydrogen and lower alkyl.

FIG. 2 shows the structure of the ribophosphoramidite monomers that areused to synthesize oligoribonucleotide N3′→P5′ phosphoramidates where: Bis cytosine, uracil, 2,6-diaminopurine, or guanine; MMTNH is(monomethoxytrityl)amino; OTBDMS is —O-t-butyldimethysilyl; iPr₂N isdiisopropylamino; and CEO is β-cyanoethyl. In addition, when B iscytosine, the N4 amino group of cytosine is protected with a benzoylgroup; when B is 2,6-diaminopurine, the exocylic amine groups areprotected with a phenoxyacetyl group, or when B is guanine the N2 aminogroup of guanine is protected with an isobutyl group.

FIG. 3 shows the overall synthetic scheme used to prepare most of theprotected ribophosphoramidite monomers of the present invention. Brepresents a base selected from the group consisting of adenine (A),guanine (G), 2,6-diaminopurine (D), uracil (U), cytosine (C) andthymidine (T). Tol is toluoyl, MMTNH is (monomethoxytrityl)amino, OTBDMSis —O-t-butyldimethysilyl, IPr₂N is diisopropylamino, and CEO is3-cyanoethyl, R is anisoyl when the base is G or D, and toluoyl when thebase is A, T, or U. In addition, when B is adenine, the N6 amino groupof adenine is protected with a benzoyl group; when B is2,6-diaminopurine, the exocylic amine groups are protected with aphenoxyacetyl group; or when B is guanine the N2 amino group of guanineis protected with an isobutyl group.

FIG. 4 shows the scheme used to prepare N⁴-benzoyl-3′-aminocytidineanalogue (10c) and5′-(2-cyanoethyl-N,N′-diisopropylamino)phosphoramidite cytidine monomer(11c). Tol is toluoyl, MMTNH is (monomethoxytrityl)amino, OTBDMS is—O-t-butyldimethysilyl, iPr₂N is diisopropylamino, and CEO isβ-cyanoethyl.

FIG. 5 shows schematics of exemplary synthesis procedures for thecompounds of the invention.). In FIG. 5, the following abbreviationsapply:

-   -   i=Cl—C(O)—R″/(i-Pr)2NEt, or HO—C(O)—R″/C.A, or        [C(O)—R″]2O/(i-Pr)2NEt    -   iv=R″—HC═O+[H]    -   R=5′-CPG-Supported P,N-Protected Oligonucleotide    -   R′=Deprotected NP- or NPS-Oligonucleotide    -   R″=lipid moiety, L (to which a linker may be conjugated, if        desired, see R′″ for an example of a conjugated amino glycerol        linker)    -   R′″═—O—CH2(CHOH)CH2-NHC(O)—R″    -   X═O, S; Y═H, or C(O)—R″, Z═O or NH    -   R₁═F, OR₂, wherein R₂ is H or alkyl.

FIGS. 5A and 5B show synthesis procedures that can be used for theproduction of compounds in which the lipid moiety is conjugated to the3′ terminus of the oligonucleotide. The scheme shown in FIG. 5B is areductive amination starting with a lipid aldehyde; this produces anamine linkage between the lipid group and the oligonucleotide (seeSchematic B below), in contrast to the scheme shown in FIG. 5A where thestarting materials are carboxylic acid, acid anhydride or acid chlorideforms of a fatty acid, resulting in the formation of an amide linkage(see Schematic A below

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

An “alkyl group” refers to a straight, branched, or cyclic, substitutedor unsubstituted alkyl group having 1 to 20 carbon atoms, such asmethyl, ethyl, propyl, and the like. Lower alkyl typically refers to C₁to C₅. Intermediate alkyl typically refers to C₆ to C₁₀. Thesubstituents can be chosen from, by way of non-limiting example,halogen, hydroxy, alkoxy, alkenyl, alkynyl, thio, nitro, amino, amide,acyl, and carboxyl.

An “acyl group” refers to a group having the structure RCO wherein R isan alkyl group. A lower acyl is an acyl wherein R is a lower alkylgroup.

An “alkylamine” group refers to an alkyl group containing at least oneattached nitrogen, and includes mono- and di-alkyl amines, e.g.,1-methyl-1-butylamine (CH₃CHNH₂CH₂CH₂CH₃), and the alkyl group can befurther substituted with at least one substituent chosen from, by way ofnon-limiting example, halogen, hydroxy, alkoxy, alkenyl, alkynyl, thio,nitro, amino, amide, acyl, and carboxyl.

An “aryl group” refers to an aromatic ring group having 5-20 carbonatoms, such as phenyl, naphthyl, anthryl, or substituted aryl groups,such as, alkyl- or aryl-substitutions like tolyl, ethylphenyl,biphenylyl, etc. Also included are heterocyclic aromatic ring groupshaving 5-20 carbon atoms and at least one, for example 1-10, nitrogen,oxygen, and/or sulfur atoms in the ring.

“Oligonucleotide” refers to ribose and/or deoxyribose nucleoside subunitpolymers having between about 2 and about 200 contiguous subunits. Thenucleoside subunits can be joined by a variety of intersubunit linkages,including, but not limited to, phosphodiester, phosphotriester,methylphosphonate, P3′→N5′ phosphoramidate, N3′→P5′ phosphoramidate,N3′→P5′ thiophosphoramidate, and phosphorothioate linkages. Further,“oligonucleotides” includes modifications, known to one skilled in theart, to the sugar (e.g., 2′ substitutions), the base (see the definitionof “nucleoside” below), and the 3′ and 5′ termini. In embodiments wherethe oligonucleotide moiety includes a plurality of intersubunitlinkages, each linkage can be formed using the same chemistry or amixture of linkage chemistries can be used. The term “polynucleotide”,as used herein, has the same meaning as “oligonucleotide” and is usedinterchangeably with “oligonucleotide”.

Whenever an oligonucleotide is represented by a sequence of letters,such as “ATGUCCTG,” it will be understood that the nucleotides are in5′→3′ order from left to right. Representation of the base sequence ofthe oligonucleotide in this manner does not imply the use of anyparticular type of internucleoside subunit in the oligonucleotide.

As used herein, “nucleoside” includes the natural nucleosides, including2′-deoxy and 2′-hydroxyl forms, e.g., as described in Komberg and Baker,DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992), and analogs.

“Analogs” in reference to nucleosides includes synthetic nucleosideshaving modified nucleobase moieties (see definition of “nucleobase”below) and/or modified sugar moieties, e.g., described generally byScheit, Nucleotide Analogs (John Wiley, New York, 1980). Such analogsinclude synthetic nucleosides designed to enhance binding properties,e.g., stability, specificity, or the like, such as disclosed by Uhlmannand Peyman (Chemical Reviews, 90:543-584, 1990).

The term “lipid” is used broadly herein to encompass substances that aresoluble in organic solvents, but sparingly soluble, if at all, in water.The term lipid includes, but is not limited to, hydrocarbons, oils, fats(such as fatty acids, glycerides), sterols, steroids, and derivativeforms of these compounds. Suitable lipids include fatty acids and theirderivatives, hydrocarbons and their derivatives, and sterols, such ascholesterol. As used herein, the term lipid also includes amphipathiccompounds, which contain both lipid and hydrophilic moieties.

Fatty acids usually contain even numbers of carbon atoms in a straightchain (commonly 12-24 carbons) and can be saturated or unsaturated, andcan contain, or be modified to contain, a variety of substituent groups.For simplicity, the term “fatty acid” also encompasses fatty acidderivatives, such as fatty amides produced by the synthesis scheme shownin FIG. 5A (see for example, the compounds shown FIGS. 1A-1E).

The term “hydrocarbon” as used herein encompasses compounds that consistonly of hydrogen and carbon, joined by covalent bonds. The termencompasses open chain (aliphatic) hydrocarbons, including straightchain and branched hydrocarbons, and saturated as well as mono- andpolyunsaturated hydrocarbons. The term also encompasses hydrocarbonscontaining one or more aromatic rings.

The term “substituted” refers to a compound that has been modified bythe exchange of one atom for another. In accordance with one aspect ofthe disclosure, the term is used in reference to halogenatedhydrocarbons and fatty acids, including those in which one or morehydrogen atoms are substituted with fluorine.

A “nucleobase” as used herein includes (i) typical DNA and RNAnucleobases (uracil, thymine, adenine, guanine, and cytosine), (ii)modified nucleobases or nucleobase analogs (e.g., 5-methyl-cytosine,5-bromouracil, or inosine), and (ill) nucleobase analogs. A nucleobaseanalog is a chemical whose molecular structure mimics that of a typicalDNA or RNA base.

As used herein, “pyrimidine” means the pyrimidines occurring in naturalnucleosides, including cytosine, thymine, and uracil, and analogsthereof, such as those containing substituents chosen from, for example,oxy, methyl, propynyl, methoxy, hydroxyl, amino, thio, and halo. Theterm as used herein further includes pyrimidines with protection groupsattached, such as N₄-benzoylcytosine. Further pyrimidine protectiongroups are disclosed by Beaucage and Iyer (Tetrahedron 48:223-2311,1992).

As used herein, “purine” means the purines occurring in naturalnucleosides, including adenine, guanine, and hypoxanthine, and analogsthereof, such as those containing substituents chosen from, for example,oxy, methyl, propynyl, methoxy, hydroxyl, amino, thio, and halo. Theterm as used herein further includes purines with protection groupsattached, such as N₂-benzoylguanine, N₂-isobutyrylguanine,N₆-benzoyladenine, and the like. Further purine protection groups aredisclosed by Beaucage and Iyer (cited above).

As used herein, the term “protected” as a component of a chemical namerefers to art-recognized protection groups for a particular moiety of acompound, e.g., “5′-protected-hydroxyl” in reference to a nucleosideincludes triphenylmethyl (i.e., trityl), p-anisyldiphenylmethyl (i.e.,monomethoxytrityl or MMT), di-p-anisylphenylmethyl (i.e.,dimethoxytrityl or DMT), and the like. Art-recognized protection groupsinclude those described in the following references: Gait, editor,Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford,1984); Amarnath and Broom, Chemical Reviews, 77:183-217, 1977; Pon etal., Biotechniques, 6:768-775, 1988; Ohtsuka et al., Nucleic AcidsResearch, 10:6553-6570, 1982; Eckstein, editor, Oligonucleotides andAnalogues: A Practical Approach (IRL Press, Oxford, 1991); Greene andWuts, Protective Groups in Organic Synthesis, Second Edition, (JohnWiley & Sons, New York, 1991); Narang, editor, Synthesis andApplications of DNA and RNA (Academic Press, New York, 1987); Beaucageand Iyer (cited above), and like references.

The term “halogen” or “halo” is used in its conventional sense to referto a chloro, bromo, fluoro or iodo substituent. In the compoundsdescribed and claimed herein, halogen substituents are generally fluoro,bromo, or chloro, suitably fluoro or chloro.

2. Design of Riboamidate and Ribothioamidate siRNAs

The riboamidate and thioriboamidate siRNAs disclosed herein includethose having the formula O-(x-L)_(n), wherein O is a riboamidate, L is alipid moiety, x is an optional linker, and n is an integer ranging from1 to 5. The design of such siRNAs requires the selection of O, L, andthe determination of the structural linkage(s) between O and L, whichmay involve the optional linker group x.

The oligonucleotide component O may be regarded as the “effector”component of the compound in that it is this component that effects RNAinterference by binding to the complementary target nucleic acidsequence. Thus, the sequence of O is chosen such that it includes aregion comprising nucleotides complementary to a target nucleic acidsequence of a gene.

The riboamidate and ribothioamidate siRNAs may be provided in singlestranded and double stranded forms. Single stranded forms are antisense(complementary to the coding strand of the targeted message), and it isthe antisense strand that is most important in the applicationsdisclosed herein.

The choice of the type of inter-nucleoside linkages used in synthesizingthe riboamidates and ribothioamidates may be made from any of theavailable oligonucleotide chemistries. For the design of the antisensestrands of single stranded and double stranded siRNAs, theinter-nucleoside linkages will generally be comprised of linkages chosenfrom N3′→P5′ phosphoramidate and N3′→P5′ thiophosphoramidate linkages.For double-stranded designs, more flexibility is permitted in thechemistry for the sense strand of double-stranded siRNAs, so that thelinkages may be chosen from, but not limited to, phosphodiester,phosphotriester, methylphosphonate, P3′→N5′ phosphoramidate, N3′→P5′phosphoramidate, N3′→P5′ thiophosphoramidate, and phosphorothioatelinkages.

Thus, according one aspect of the present invention, the siRNAsdisclosed herein contain at least one linkage chosen from N3′→P5′phosphoramidate, and N3′→P5′ thiophosphoramidate linkages, which may berepresented by the structure: 3′-[—NH—P(═O)(—XR)—O—]-5′, wherein X is Oor S and R is chosen from hydrogen, alkyl, and aryl; andpharmaceutically acceptable salts thereof. According to another aspectof the invention, the linkages of the siRNAs disclosed herein arecomprised entirely of N3′→P5′ phosphoramidate and/or N3′→P5′thiophosphoramidate linkages. According to yet another aspect of theinvention, at least 60% of the total linkages of the siRNAs disclosedherein are comprised of N3′→P5′ phosphoramidate and/or N3′→P5′thiophosphoramidate linkages. In accordance with another aspect of theinvention, at least 70%, for example at least 80% of the total linkagesof the siRNAs disclosed herein are N3′→P5′ phosphoramidate and/orN3′→P5′ thiophosphoramidate linkages.

According to one aspect of the invention herein, the nucleotides of thesiRNAs are comprised entirely of RNA nucleosides. According to anotheraspect, the siRNAs are comprised of RNA and DNA nucleosides. Thus, theterm siRNA as used herein is intended to encompass chimeric molecules inwhich, while the majority of the nucleotides are RNA, it is permissiblefor some of the nucleotides to be DNA. This is especially the case forthe sense region of double-stranded siRNAs.

Where a lipid moiety is to be conjugated to the 3′ terminus of theriboamidate and ribothioamidate siRNAs disclosed herein, the synthesisof the conjugate is greatly facilitated by the presence of a 3′ aminogroup. Hence, and irrespective of the chemistry selected, the additionof a 3′ amino group is advantageous. The siRNAs are typically at least17 bases in length, up to 50 bases in length, more usually from about 19to about 25 bases in length. The siRNAs disclosed herein comprise asequence of nucleotides complementary to a target nucleic acid sequence.According to one aspect of the invention, the nucleotide sequence of thesiRNA is exactly complementary to the target nucleic acid sequence.However, it is not always necessary that the full length of the sequenceof the nucleotide component be exactly complementary to the targetsequence, the sequence can include residues or regions that are notcomplementary to the target sequence. Thus, according to another aspectof the invention, the nucleotide sequence of the siRNA is less thanexactly complementary to the target nucleic acid sequence. The degree ofcomplementarity depends on a variety of factors, such as, for example,the constitution of the gene targeted for RNA interference.

3. Synthesis of Riboamidate and Ribothioamidate siRNAs

According to one aspect of the present invention, the compounds arerepresented by the formula:O-(x-L)_(n),where O represents the amidate, x is an optional linker group, Lrepresents the lipid moiety, and n is an integer from 1-5.

Generally, the riboamidates and ribothioamidates disclosed herein can beprepared by a process comprising:

-   -   1) providing a first 3′-amino protected nucleoside, which is        optionally attached to a solid phase support;    -   2) deprotecting the protected 3′-amino group to form a free        3′-amino group;    -   3) reacting the free 3′-amino group with a protected        phosphoamidite ribonucleoside monomer to form an internucleoside        N3′→P5′ phosphoramidate linkage; and    -   4) oxidizing (or sulfurizing) the internucleoside N3′→P5′        phosphoramidite linkage to form a phosphoramidate (or        thiophosphoramidate) linkage.

According to one aspect of the invention, the protected phosphoramidateribonucleoside monomers are(2′-t-butyldimethylsilyl)-3′-(monomethoxytrityl)-amino-5′-O-(cyanoethyl-N,N′-diisopropyl-amino)-phosphoramitenucleoside monomers. In addition, the method of synthesizing anoligoribonucleotide can further include capping the free 3′ amino groupsthat fail to react with the protected phosphoramidite ribonucleosidemonomer.

Also disclosed herein is a solid phase method of synthesizingoligonucleotide N3′→P5′ thiophosphoramidates using a modification of thephosphoramidite transfer methodology of Gryaznov, Tetrahedron Letters,7661-64 (1999). Suitable non-limiting examples of solid phase supportsinclude glass, beads, silica, etc. The synthetic strategy employed3′-NH-trityl-protected 3′-aminonucleoside5′-O-cyanoethyl-N,N-diisopropylaminophosphoramidites that were made bythe method described in detail below. Every synthetic cycle wasconducted using the following chemical procedures: 1) detritylation, 2)coupling; 3) capping; and 4) sulfurization. For a step-wisesulfurization of the internucleaside phosphoramidite group formed afterthe coupling step, the iodine/water based oxidizing agent was replacedby the sulfurizing agents—either by elemental sulfur S₈ or by thecommonly used Beaucage reagent—3H-1,2-benzodithiol-3-one 1,1 dioxide(Iyer et al., J. Organic Chemistry 55:4693-4699, 1990). Theoligonucleotide syntheses were performed (1 μmole synthesis scale) witha 1% solution of Beaucage reagent in anhydrous acetonitrile or 15% S₈ inCS₂/Et₃N, 99/1 (vol/vol) as the sulfurizing agent.

Chimeric N3′→P5′ phosphoramidate-phosphorthioamidate oligonucleotidescan be made by using an oxidation step(s) after the coupling step, whichresults in formation of a phosphoramidate internucleoside group.Similarly, phosphodiester-phosphorthioamidates can be made by using5′-phosphoramidite-3′-O-DMTr-protected nucleotides as monomeric buildingblocks.

Initial investigations into the assembly of oligoribonucleotide N3′→P5′phosphoramidates determined that a synthetic methodology based on aphosphoramidite transfer reaction was suitable for construction of thesebiopolymers (Gryaznov, et al. (1998) Nucleic Acids Res., 26:4160-4167).This approach was previously employed for the synthesis ofoligo-2′-fluoro-2′-deoxynucleotide N3′→P5′ phosphoramidates as well asfor oligo-2′-deoxynucleotide N3′→P5′ phosphoramidates (Schultz, et al.(1996) Nucleic Acids Res., 24, 2966-2973; McCurdy, et al. (1997)Tetrahedron Lett., 38, 207-210; Nelson, et al. (1997) J. Org. Chem., 62,7278-7287). The synthetic strategy employed in accordance with thepresent invention is based on the use of3′-(monomethoxytrityl)amino-5′-O-(cyanoethyl-N,N′-diisopropylamino)-phosphoramiditenucleoside monomers (FIG. 2) and assembly of the oligoribonucleotide inthe 5′ to 3′ direction. The appropriately protected ribonucleotidemonomers were in general synthesized according to the previouslyreported protocols (Gryaznov, et al. (1998) Nucleic Acids Res.,26:4160-4167), which were modified thereby allowing for maximization ofoverall yields and expediting isolation of the final products (FIG. 3).

Disclosed herein is a synthetic method for the preparation of themonomers, resulting in the rapid access to the final products withimproved overall yields. In general, the 2′ position is selectivelydeprotected; the azido group at the 3′ position is reduced to an amine;the 2′ and 3′ position are then protected, suitably with each positionhaving a different protecting group such that each position can beselectively deprotected; the 5′ protecting group is selectivelydeprotected; and the 5′-OH group is phosphitylated to provide themonomers of FIG. 2 that are the phosphoramide building blocks.

The selective removal of the protecting group at the 2′ position and thereduction of the azido group at the 3′ position can be done sequentiallyor concurrently if the protecting group is chosen such that it can beremoved under the reduction conditions. Thus, if the 2′ position isprotected with a benzyl group, and the reduction is done under theappropriate conditions, the removal of the benzyl group and thereduction of the azido group can be accomplished in one step. Generally,the 2′ position is deprotected to prevent the commonly used 2′protecting groups, such as acetyl and benzoyl, from migrating to the 3′amino position. Thus, according to one aspect of the invention, theprotecting group at the 2′ position is chosen such that it does notmigrate to the 3′ position and therefore does not need to be removedbefore reduction of the azido group.

As depicted in FIG. 3, the first step of the synthesis involved tin(IV)chloride or trimethylsilyl triflate mediated glycosylation oftrimethylsilylated nucleobases (Azhayev, et al. (1979) Nucleic AcidsRes., 2:2625-2643; Vorbruggen, et al. (1981) Chem. Ber., 114:1234-1255)to a commonly employed sugar precursor3-azido-1,2-di-O-acetyl-5-O-toluoyl-3-deoxy-D-ribofuranose 1, which wasprepared according to literature procedure (Ozols, et al. Synthesis,557-558). Adenine was protected at N⁶ with a benzoyl group, whileguanine was blocked at N² with an isobutyl group and at O⁶ withdiphenylcarbamate (Zou, et al. (1987) Can. J. Chem., 65:1436-1437). Theprotection of O⁶ with this bulky group allows for selectiveglycosylation to occur at N⁹ with very little (≦10%) formation of theundesired N⁷ regioisomer as judged by TLC analysis. 2,6-Diaminopurinewas protected at each exocylic amine with a phenoxyacetyl group for allglycosylation reactions with this highly polar purine base analogue(Schulhof, et al. (1987) Tetrahedron Left., 28:51-54).

Then experimental conditions were found, which provided for theselective removal of the 2′-O-protecting group, such as acetyl, benzyl,benzoyl, or trialkylsilyl, in the presence of the 5′-O-protecting group,such as toluoyl or benzoyl (Neilson, et al. (1971) Can. J. Chem.,49:493-498) (FIG. 3). This allowed for the omission of a 5′-hydroxylreprotection step from the synthetic protocol. Also, a low yieldingseries of steps late in the monomer synthesis, used in the literatureprocedure (Gryaznov, et al. (1998) Nucleic Acids Res., 26:4160-4167) toconvert a 5′-O-trityl-nucleoside precursor to the 3′-N-trityl-protectedamino intermediate, were also avoided.

Following the glycosylation reaction, the next five chemicaltransformations resulted in very high yields of the products. Thiseliminated the need for intermediate purification after the chemicalconversions of iv-vii. (FIG. 3), thus providing a rapid and convenientaccess to compounds of structure 8. However, it should be noted that forthe guanosine and 2,6-diaminopurine analogues, selective removal of the2′-O-acetyl protecting group was unsuccessful. Thus, both 2′-O- and5′-O-protecting groups were removed, after which the 5′-hydroxyl groupwas selectively reprotected (FIG. 3(iii)).

For compound 2 (FIG. 3), where the base (B) was A, T, or U, the2′-O-acetyl group was selectively removed using a base, optionally in anhydrophilic organic solvent followed by the reduction of the 3′-azidogroup to an amine group.

Appropriate solvents are those which will at least partially dissolveone or all of the reactants and will not adversely interact with eitherthe reactants or the product. Non-limiting examples of suitable solventsinclude aromatic hydrocarbons such as toluene, o-, m- and p-xylene,halogenated hydrocarbons, such as methylene chloride, chloroform andchlorobenzene, ethers such as diethyl ether, diisopropyl ether,tert-butyl methyl ether, dioxane, anisole, and tetrahydrofuran,nitriles, such as acetonitnle and propionitrile, ketones, such asacetone, methyl ethyl ketone, diethyl ketone, and tert-butyl methylketone, alcohols such as methanol, ethanol, n-propanol, isopropanol,n-butanol and tert-butanol, and also dimethyl sulfoxide (DMSO),dimethylformamide (DMF), and water. According to one aspect, thesuitable solvents are chosen from DMSO, DMF, acetonitrile, and toluene.Mixtures of solvents can also be used.

Non-limiting examples of suitable bases include, generally, inorganiccompounds, such as alkali metal hydroxides and alkaline earth metalhydroxides, such as lithium hydroxide, sodium hydroxide, potassiumhydroxide, and calcium hydroxide, alkali metal oxides and alkaline earthmetal oxides, such as lithium oxide, sodium oxide, calcium oxide, andmagnesium oxide, alkali metal hydrides and alkaline earth metalhydrides, such as lithium hydride, sodium hydride, potassium hydride andcalcium hydride, alkali metal amides, such as lithium amide, sodiumamide, and potassium amide, alkali metal carbonates and alkaline earthmetal carbonates, such as lithium carbonate and calcium carbonate, andalso alkali metal hydrogen carbonates such as sodium hydrogen carbonate,organometal compounds, in particular alkali metal alkyls, such asmethyllithium, butyllithium, and phenyllithium, alkylmagnesium halides,such as methylmagnesium chloride, and alkali metal alkoxides andalkaline earth metal alkoxides, such as sodium methoxide, sodiumethoxide, potassium ethoxide, potassium tert-butoxide, anddimethoxymagnesium, furthermore organic bases, e.g. tertiary amines,such as trimethylamine, triethylamine, tri-isopropylamine andN-methylpiperidine, pyridine, substituted pyridines, such as collidine,lutidine, and 4-dimethylaminopyridine, and also bicyclic amines.According to one aspect of the invention, the suitable base comprises atleast one of sodium hydride, potassium hydroxide, potassium carbonateand triethylamine. According to another aspect, 50% (v/v) aqueousammonia in methanol is used.

The azido group in the compounds of FIG. 3 can be reduced to an aminegroup by hydrogenation. Typically, hydrogenation is carried out using anoble metal catalyst, such as palladium, platinum, rhodium, or the like,optionally on carbon, as is well known in the art. Each of thesereactions proceeded with very high, near quantitative, yields as judgedby TLC and ¹H NMR analysis of the products. The obtained nucleosideprecursors were then sequentially protected at the 2′-hydroxyl with atrialkylsilyl containing group and at the 3′-amino group with asubstituted or unsubstituted trityl group to give compound 7 (FIG. 3).After workup, the crude mixtures were treated with a base, such as 1 Msolution of sodium hydroxide in pyridine/methanol/water, to remove the5′-O-toluoyl group and afford nucleoside 8 with overall yields of56%-60% based on starting precursors 2 (FIG. 3).

For compound 2, where the base is G or D and the 2′-O-position isprotected with the acetyl group, the 2′-O-acetyl group could not beselectively removed. Therefore, in an alternative scheme, both 2′-O- and5′-O-protecting groups were removed using a base, such as 1 M sodiumhydroxide, after which a 5′-O-anisoyl group was selectively reintroducedunder Mitsunobu conditions to give 4. It should be noted that the highreactivity of the 2′-hydroxyl group of the 3′-azido-2′-hydroxylguanosine intermediate prevented selective reprotection of the5′-hydroxyl group by either benzoyl chloride or benzoyl anhydride. Thesame series of steps described above was then used to convert 4 where Bis G or D into the corresponding compound 8. The final step for monomerpreparation involved phosphitylation of compound 8 to give the5′-(2-cyanoethyl-N,N′-diisopropylamino)nucleoside phosphoramiditebuilding blocks 9 (FIG. 3).

In an alternative synthetic transformation, the intermediate compound 7,where B is uridine, was converted into a N⁴-benzoyl-3′-aminocytidineanalogue (10). Initially, the uridine derivative 7 was transformed intothe benzoyl protected cytosine derivative according to literatureprocedure (FIG. 4) (Divacar, et al. (1982) J. Chem. Soc. Perkin Trans.,1:1171-1176). Reaction of 7u (base is U) with triazole in the presenceof phosphorus oxychloride yielded the desired 4-triazolo species, whichupon treatment with ammonia generated the 4-amino-unprotected cytosinenucleoside. After workup, the crude reaction mixture was benzoylated andfinally deprotected with 1 M sodium hydroxide to give 10c (FIG. 4) in45% overall yield from the starting compound 7. Phosphitylation of 10cproduced the desired5′-(2-cyanoethyl-N,N′-diisopropylamino)phosphoramidite cytidine monomerused for oligonucleotide construction.

In an alternative method, the appropriately protected2′-O-alkyl-3′aminonucleoside-5′-phosphoramidite building blocks 4, 6,11, and 15, where alkyl is methyl, were prepared according to a seriesof chemical transformations shown in Schemes 1-3 below. A step for thepreparation of these compounds was the selective methylation of the2′-hydroxyl group in the presence of either the imino functionality ofpyrimidines, or the N-7 atom of the purines. The two pyrimidine-basedmonomers were obtained from the known3-azido-2′-O-acetyl-5′-O-toluoyl-3′-deoxy-3-D-ribofuranosyluracil 1.Typically, the N-3/O-4 imino nitrogen of 1 was first protected with aprotecting group, such as by the reaction of methyl propyolate in thepresence of dimethylaminopyridine (Scheme 1). The crude reaction productwas then selectively 2′-O-deacetylated, and the resulting free2′-hydroxyl group was then alkylated, such as by methylation usingiodomethane and silver oxide. The N-3 protecting group was removed andthe 3′-azido group was reduced to amine, which was then immediatelyprotected, such as reaction with 4-monomethoxytritylchloride, to givethe precursor 3. The 5′-toluoyl ester was then cleaved using an alkalinesolution, followed by phosphitylation using known protocols to give thedesired 2′-O-methyl uridine phosphoramidite monomer 4. The 2′-O-methylcytosine phosphoramidite was obtained by conversion of uridineintermediate 3 into 3′-aminocytidine analogue 5.

The synthesis of the 2′-O-alkyl adenosine analogue required the use ofbulky protecting groups, primarily for exocyclic amine in order toprevent the alkylation of N-7 during methylation of the 2′-hydroxylgroup (Scheme 2).3′-Azido-2′-O-acetyl-5′-O-toluoyl-N⁶-benzoyl-3′-deoxyadenosine 7 wasfirst deprotected, such as by reaction with NH₃/MeOH (1/1, v/v), toafford 3′-azido-3′-deoxyadenosine. Then, the 5′-hydroxyl group and theN-6 moiety were selectively re-protected with bulky protecting groups,such as the t-butyldiphenylsilyl group or the 4-monomethoxytrityl group.The combination of the two large substituents at the 5′-0 and N-6positions sterically occluded N-7, thereby allowing for the selective

introduction of a methyl group at the 2′-position to produce theintermediate 8. The N-6 4-monomethoxytrityl group was then removed, suchas by treatment with 3% trichloroacetic acid in an organic solvent, suchas dichloromethane, followed by re-protection of N-6. The use of benzoylchloride for the re-protection of N-6 resulted in the addition of twobenzoyl groups. The second benzoyl group was subsequently removed bybase treatment to produce the intermediate 9. The azide group was thenreduced and the resulting 3′-amino group was protected with4-monomethoxytrityl to form 10. Finally, the 5′-silyl protecting groupwas cleaved, and phosphitylation resulted in the 2′-O-methylphosporamidite monomer 11.

The synthesis of the guanosine-based 2′-O-alkyl phosphramidite 15 isdepicted in Scheme 3.3′-Azido-2′-O-acetyl-5′-O-toluoyl-N²-isobutryl-O⁶-diphenylcarbamoyl-3′-deoxyguanosine12 was deblocked by treatment with a base. The 5′O- and O-6 werereprotected by reaction with t-butyldiphenylsilylchloride. Thebis-silylated intermediate was then 2′-O alkylated. The O-6 silyl groupwas selectively deprotected to give compound 13. The N-2 group wasre-protected, the 3′-azido group was reduced, and the resulting 3′-aminogroup was protected to yield the nucleoside 14. Finally, the 2′-O-alkylguanosine phosphoramidite monomer 15 was obtained by removing the5′-protecting group followed by phosphitylation of the unmasked5′-hydroxyl.

The double-stranded form of the siRNAs can be prepared by synthesizingthe two single strands and adding one to the other by, e.g., annealingthe strands. It is also possible to prepare a double-stranded form ofthe siRNA by constructing a single strand and allowing it to fold uponitself and form a hairpin duplex.

4. Design of Lipidated Riboamidate and Ribothioamidate siRNAs

The riboamidate and thioriboamidate siRNAs conjugated to lipidcomponents are effective in RNAi applications, such as therapeutic RNAiapplications, possibly more so than corresponding unconjugatedriboamidates and ribothioamidates. The lipid component L is believed tofunction to enhance cellular uptake of the siRNA, particularly infacilitating passage through the cellular membrane. While the mechanismby which this occurs has not been fully elucidated, one possibility isthat the lipid component may facilitate binding of the siRNA to the cellmembrane as either a single molecule, or an aggregate (micellar) form,with subsequent internalization. However, understanding of the precisemechanism is not required for the invention to be utilized.

The lipid component can be any lipid or lipid derivative that providesenhanced cellular uptake compared to the unmodified riboamidate orribothioamidate. Suitable non-limiting examples of lipids useful inaccordance with the present invention include hydrocarbons, fats (e.g.,glycerides, fatty acids and fatty acid derivatives, such as fattyamides) and sterols. Where the lipid component is a hydrocarbon, the Lcomponent can be a substituted or unsubstituted cyclic hydrocarbon or analiphatic straight chain or branched hydrocarbon, which can be saturatedor unsaturated. Suitable examples include straight chain unbranchedhydrocarbons that are fully saturated or polyunsaturated. The length ofthe hydrocarbon chain can vary from C₂-C₃₀, but optimal results can beobtained with carbon chains that are C₈-C₂₂. Suitable non-limitingexamples of saturated hydrocarbons (alkanes) are listed below:

Systematic name Carbon chain Tetradecane C₁₄H₃₀ Pentadecane C₁₅H₃₂Hexadecane C₁₆H₃₄ Heptadecane C₁₇H₃₆ Octadecane C₁₈H₃₈ Nonadecane C₁₉H₄₀Eicosane C₂₀H₄₂

Mono- and poly-unsaturated forms (alkenes and polyenes, such asalkadienes and alkatrienes) of hydrocarbons can also be selected, withcompounds having one to three double bonds being suitable examples,although compounds having more double bonds can be employed. Alkynes(containing one or more triple bonds) and alkenynes (triple bond(s) anddouble bond(s)) can also be utilized. Examples of common mono- andpoly-unsaturated hydrocarbons that can be employed include those shownin FIGS. 1M, 1L and 1O.

Substituted forms of hydrocarbons can be employed in the compounds ofthe invention, with substituent groups that are inert in vivo and invitro being suitable. An example of such a suitable substituent isfluorine. Exemplary generic structures of polyfluorinated hydrocarbonsinclude:CF₃(CF₂)_(n)—(CH₂)_(m)—where m is at least 1, for example at least 2, and n=1-30, such asfluorotridecane:CF₃(CF₂)₉(CH₂)₃; andCH₃(CH₂)_(a)(CF₂)_(b)(CH₂)_(c)—where a, b and c are independently 1-30.

FIG. 1W shows an example of a polyfluorinated hydrocarbon conjugated tothe 5′ terminus of an oligonucleotide.

Other suitable lipid components include simple fatty acids and fattyacid derivatives, glycerides, and more complex lipids such as sterols,for example cholesterol. Fatty acids and their derivatives can be fullysaturated or mono- or poly-unsaturated. The length of the carbon chaincan vary from C₂-C₃₀, but optimal telomerase inhibition can be obtainedwith carbon chains that are C₈-C₂₂. Suitable non-limiting examples ofsaturated fatty acids are listed below:

Systematic name Trivial name Carbon chain Tetradecanoic myristic 14:0Hexadecanoic palmitic 16:0 Octadecanoic stearic 18:0 Eicosanoicarachidic 20:0

Mono- and poly-unsaturated forms of fatty acids can also be employed,with compounds having one to three double bonds being suitable examples,although compounds having more double bonds can also be employed.Examples of common mono- and poly-unsaturated fatty acids that can beemployed include:

Systematic name Trivial name Carbon chain Cis-9-hexadecanoic palmitoleic16:1(n-7) Cis-6-octadecanoic petroselinic 18:1 (n-12) Cis-9-octadecanoicoleic 18:1 (n-9) 9,12-octadecadienoic linoleic 18:2 (n-6)6,9,12-octadecatrienoic gamma-linolenic 18:3 (n-6)9,12,15-octadecatrienoic alpha-linolenic 18:3 (n-3)5,8,11,14-eicosatetraenoic arachidonic 20:4 (n-6)

Fatty acids with one or more triple bonds in the carbon chain, as wellas branched fatty acids can also be employed in the compounds disclosedherein. Substituted forms of fatty acids can be employed in thecompounds disclosed herein. As with the hydrocarbon groups, substituentgroups that are inert in vivo and in vitro are suitable examples, withfluorine being an example of such a group. Exemplary generic structuresof polyfluorinated derivatives of fatty acids suitable for use inaccordance with the present invention are:CF₃(CF₂)_(n)(CH₂)_(m)CO—where m is at least 1, for example at least 2, and n=1-30, andCH₃(CH₂)_(a)(CF₂)_(b)(CH₂)_(c)CO—where a, b and c are independently 1-30

Examples of compounds having polyfluorinated derivatives of fatty acidsare shown in FIGS. 1U and 1V.

Typically, between one and five L components (n=1-5) are covalentlylinked to the O component, optionally via a linker. For example, one ortwo L components are utilized (n=1 or 2). Where more than one Lcomponent is linked to the O component, each L component isindependently selected.

It will be appreciated that compounds described as having a specifiedhydrocarbon as the L moiety and compounds described as having aspecified fatty acid (with the same number of carbon atoms as thespecified hydrocarbon) are closely related and differ in structure onlyin the nature of the bond that joins the L moiety to the riboamidate orribothioamidate, which in turn is a result of the synthesis procedureused to produce the compound.

For example, and as described in more detail below, when compounds aresynthesized having the L moiety conjugated to the 3′-amino terminus of ariboamidate (having phosphoramidate or thiophosphoramidateinternucleoside linkages), the use of the aldehyde form of a fatty acid(a fatty aldehyde) as the starting material results in the formation ofan amine linkage between the lipid chain and the riboamidate, such thatthe lipid group appears as a hydrocarbon. In contrast, use of thecarboxylic acid, acid anhydride or acid chloride forms of the same fattyacid results in the formation of an amide linkage, such that the lipidgroup appears as a fatty acid derivative, specifically in this instancea fatty amide (as noted in the definitions section above, for the sakeof simplicity, the term “fatty acid” when describing the conjugated Lgroup is used broadly herein to include fatty acid derivatives,including fatty amides).

This is illustrated in the following schematics (and in FIGS. 5A and5B), which depict the 3′-amino terminus of a phosphoramidateoligonucleotide joined to a C₁₄ lipid component. In schematic A, L istetradecanoic acid (myristic acid), in which the connection between Land O groups is an amide. In schematic B, L is tetradecane, and theconnection between the L and O groups is an amine.

The linkage between the O and L components can be a direct linkage, orcan be via an optional linker moiety, x. The linker group can serve tofacilitate the chemical synthesis of the compounds (discussed in thesynthesis section below). Whether or not a linker group is used tomediate the conjugation of the O and L components, there are multiplesites on the riboamidate component O to which the L component(s) can beconveniently conjugated. Suitable linkage points include the 5′ and 3′termini, one or more sugar rings, the internucleoside backbone and thenucleobases of the riboamidates. Typically, the L moiety is attached tothe 3′ or 5′ terminus of the riboamidate.

If the L component is to be attached to the 3′ terminus, the attachmentcan be directly to the 3′ substituent, which in the case of thephosphoramidate and thiophosphoramidate riboamidates is the 3′-aminogroup (examples are shown in FIGS. 1A-C), and in other instances, suchas conventional phosphodiester oligonucleotides, is a 3-hydroxy group.Alternatively, the L moiety can be linked via a 3′-linked phosphategroup (an example is shown in FIG. 1Z, in which a hexadecane hydrocarbonis linked to the 3′ phosphate of a thiophosphoramidate oligonucleotidethrough an O-alkyl linker. If the L moiety is to be linked to the 5′terminus, it is typically attached through a 5′-linked phosphate group(see FIG. 1F which shows the use of an amino glycerol linker, and FIG.1G which shows the use of a bis-amino glycerol linker). Attachment to abase on the O moiety can be through any suitable atom, for example tothe N² amino group of guanosine (see FIGS. 1Q-R). Where n>1 such that aplurality of lipid moieties is to be attached to the O component, theindividually selected L components can be attached at any suitablesite(s). For example, one L group can be attached to each terminus,various L groups can be attached to the bases, or two or more L groupscan be attached at one terminus (see FIGS. 1E, 1J, 1K).

In the case of single-stranded (antisense) siRNA, the lipid is suitablyconjugated to the 3′ end, as the presence of the 5-OH is believed to beimportant to the activity of the siRNA. In the case of double-strandedsiRNAs, if the lipid is conjugated to the sense strand, the conjugationcan be at either end. If the lipid is conjugated to the antisensestrand, then it is suitably conjugated at the 3′ end, or at the base.For double-stranded siRNAs, it is possible for more than one lipid to beconjugated to the siRNA.

The optional linker component x can be used to join the O and Lcomponents of the compounds. If a linker is to be employed, it isincorporated into the synthesis procedures as described in the briefdescription of FIG. 5, above. Examples of suitable linker groups includeamino glycerol and O-alkyl glycerol-type linkers, which, respectively,can be depicted by the generic structures:

Wherein R′═H, OH, NH₂ or SH; Y═O, S or NR; R═H or alkyl; and n and m areindependently integers between 1-18.

Specific examples of suitable linkers are the aminoglycerol linker inwhich R′═OH, Y═O, and m and n are each 1:

the bis-aminoglycerol linker, in which R′═OH, Y═NH, and m and n are each1:

and the O-alkyl glycerol linker in which R═H:

Examples of compounds disclosed herein are shown in FIG. 1. Forsimplicity, only one base of the component O is shown, with a genericbase, B, being depicted and R indicating the attachment point for theremainder of the riboamidate. Compounds linked to the 3′ terminus areillustrated with a 3′-nitrogen, consistent with thiophosphoramidate andphosphoramidate riboamidate chemistries. FIGS. 1A-1L illustratecompounds having saturated lipid groups attached to the 5′ or 3′termini. FIGS. 1M-1P illustrate compounds having mono- orpoly-unsaturated lipid groups. FIGS. 1Q-1R illustrate compounds havinglipid groups conjugated to the riboamidate through a base (in this case,guanosine). FIGS. 1S and 1CC illustrate 3′- and 5′-conjugatedcholesterol lipid moiety, respectively. FIGS. 1U and 1V illustrate5′-conjugated polyfluorine substituted fatty acid derivatives, and FIG.1W illustrates a 5′ conjugated polyfluorinated hydrocarbon. FIGS. 1X-Zillustrate 5′ lipid moieties containing oxygen. The nomenclature usedherein for each of the lipid groups illustrated is as follows:

FIG. 1A: 3′-myristoylamide

FIG. 1B: 3′-palmitoylamide

FIG. 1C: 3′-stearoylamide

FIG. 1D: 3′-palmitoylamido-propyl-thiophosphate

FIG. 1E: 3′-lysyl-bis-stearoylamide

FIG. 1F: 5′-palmitoylamido-aminoglycerol-thiophosphate

FIG. 1G: 5′-palmitoylamido-bis-aminoglycerol-thiophosphate

FIG. 1H: 5′-stearoylamido-aminoglycerol-thiophosphate

FIG. 1I: 3′-dodecyl

FIG. 1J: 3′-bis-dodecyl

FIG. 1K: 3′-bis-decyl

FIG. 1L: 3′-eicosanoylamide

FIG. 1M: 3′-oleinylamide

FIG. 1N: 3′-linolenylamide

FIG. 1O: 3′-linoleylamide

FIG. 1P: 3′-trityl

FIG. 1Q: N²-tetradecyl guanosine

FIG. 1R: N²-octadecyl-guanosine

FIG. 1S: 3′-cholesterylamido-aminoglycerol-thiophosphate

FIG. 1T: 5′-(12-OH)-stearoyl-thiophosphate

FIG. 1U: 5′-C11-Teflon-thiophosphate

FIG. 1V: 5′-C13-Teflon-thiophosphate

FIG. 1W: 5′-OH—C10-Teflon-thiophosphate

FIG. 1X: 5′-OH-palmityl-thiophosphate

FIG. 1Y: 5′-batyl-thiophosphate

FIG. 1Z: 3′-batyl-thiophosphate

FIG. 1AA: 3′-palmitoylamido-aminoglycerol-thiophosphate

FIG. 1BB: 3′-thioctylamide

FIG. 1CC: 5′-cholesterylamido-aminoglycerol-thiophosphate

FIG. 1DD: 5′-(2-OH)-hexadecanol-thiophosphate

5. Synthesis of Lipidated Riboamidate and Ribothioamidate siRNAs

A variety of synthetic approaches can be used to conjugate the lipidmoiety L to the riboamidate, depending on the nature of the linkageselected, including the approaches described in Mishra et al., (1995)Biochemica et Biophysica Acta, 1264:229-237, Shea et al., (1990) NucleicAcids Res. 18:3777-3783, and Rump et al., (1998) Bioconj. Chem.9:341-349. The synthesis of compounds in which the lipid moiety isconjugated at the 5′ or 3′ terminus of the riboamidate can be achievedthrough use of suitable functional groups at the appropriate terminus,most typically an amino group, which can be reacted with carboxylicacids, acid chlorides, anhydrides and active esters. Thiol groups arealso suitable as functional groups (see Kupihar et al., (2001)Bioorganic and Medicinal Chemistry 9:1241-1247). Both amino- andthiol-modifiers of different chain lengths are commercially availablefor riboamidate synthesis.

Riboamidates having N3′→P5′ phosphoramidate and N3′→P5′thiophosphoramidate linkages contain 3′-amine groups (rather than3′-hydroxy found in most conventional oligonucleotide chemistries), andhence these riboamidates provide a unique opportunity for conjugatinglipid groups to the 3′-end of the riboamidate.

Various approaches can be used to attach lipid groups to the termini ofriboamidates with the N3′→P5′ phosphoramidate and N3′→P5′thiophosphoramidate chemistries. Examples of synthetic schemes forproducing the conjugated compounds are shown in FIG. 5.

For attachment to the 3′ terminus, the conjugated compounds can besynthesized by reacting the free 3′-amino group of the fully protectedsolid support bound riboamidates with the corresponding acid anhydridefollowed by deprotection with ammonia and purification. Alternatively,coupling of carboxylic acids of lipids to the free 3′-amino group of thesupport bound riboamidate using coupling agents, such as carbodiimides,HBTU or 2-chloro-1-methylpyridinium iodide can be used to conjugate thelipid groups. These two methods will form an amide bond between thelipid and the riboamidate. Lipids can also be attached to theriboamidate chain using a phosphoramidite derivative of the lipidcoupled to the riboamidate during chain elongation. This approach yieldsa phosphoramidate or thiophosphoramidate linkage connecting the lipidand the riboamidate (exemplified by propyl-palmitoyl and2-hydroxy-propyl-palmitoyl compounds). Still another approach involvesreaction of the free 3′-amino group of the fully protected support boundriboamidate with a suitable lipid aldehyde, followed by reduction withsodium cyanoborohydride, which produces an amine linkage.

For attachment to the 5′ terminus of, e.g., the sense strand of adouble-stranded siRNA, the riboamidate can be synthesized using amodified, lipid-containing solid support, followed by synthesis of theriboamidate in the 5- to 3′ direction as generally described in Pongracz& Gryaznov (1999). An example of the modified support is provided inSchematic C below. In the instance where n=14, the fatty acid ispalmitic acid: reaction of 3-amino-1,2-propanediol with palmitoylchloride, followed by dimethoxytritylation and succinylation providedthe intermediate used for coupling to the solid support. R is long chainalkyl amine controlled pore glass.

6. Formulation of Invention Compounds

For therapeutic application, a compound according to the presentinvention is formulated in a therapeutically effective amount with apharmaceutically acceptable carrier. One or more such compounds (forexample, having different L or O components) can be included in anygiven formulation. The pharmaceutical carrier can be solid or liquid.Liquid carriers can be used in the preparation of solutions, emulsions,suspensions, and pressurized compositions. The compounds are dissolvedor suspended in a pharmaceutically acceptable liquid excipient. Suitableexamples of liquid carriers for parenteral administration of theriboamidate preparations include water (which can contain additives,e.g., cellulose derivatives, for example sodium carboxymethyl cellulosesolution), phosphate buffered saline solution (PBS), alcohols (includingmonohydric alcohols and polyhydric alcohols, e.g., glycols) and theirderivatives, and oils (e.g., fractionated coconut oil and arachis oil).The liquid carrier can contain other suitable pharmaceutical additivesincluding, but not limited to, the following: solubilizers, suspendingagents, emulsifiers, buffers, thickening agents, colors, viscosityregulators, preservatives, stabilizers, and osmolarity regulators.

For parenteral administration of the compounds, the carrier can also bean oily ester, such as ethyl oleate and isopropyl myristate. Sterilecarriers are useful in sterile liquid form compositions for parenteraladministration.

Sterile liquid pharmaceutical compositions, solutions or suspensions canbe utilized by, for example, intraperitoneal injection, subcutaneousinjection, intravenously, or topically. The riboamidates can also beadministered intravascularly or via a vascular stent.

The liquid carrier for pressurized compositions can be a halogenatedhydrocarbon or other pharmaceutically acceptable propellant. Suchpressurized compositions can also be lipid encapsulated for delivery viainhalation. For administration by intranasal or intrabronchialinhalation or insufflation, the riboamidates can be formulated into anaqueous or partially aqueous solution, which can then be utilized in theform of an aerosol.

The compounds can be administered topically as a solution, cream, orlotion, by formulation with pharmaceutically acceptable vehiclescontaining the active compound.

The pharmaceutical compositions can be orally administered in anyacceptable dosage including, but not limited to, formulations incapsules, tablets, powders or granules, and as suspensions or solutionsin water or non-aqueous media. Pharmaceutical compositions and/orformulations comprising the riboamidates as disclosed herein can includecarriers, lubricants, diluents, thickeners, flavoring agents,emulsifiers, dispersing aids or binders. In the case of tablets for oraluse, carriers which are commonly used include lactose and corn starch.Lubricating agents, such as magnesium stearate, are also typicallyadded. For oral administration in a capsule form, useful diluentsinclude lactose and dried corn starch. When aqueous suspensions arerequired for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents can also be added.

While the compounds have superior characteristics for cellular andtissue penetration, they can be formulated to provide even greaterbenefit, for example, in liposome carriers. The use of liposomes tofacilitate cellular uptake is described, for example, in U.S. Pat. No.4,897,355 and U.S. Pat. No. 4,394,448. Numerous publications describethe formulation and preparation of liposomes. The compounds can also beformulated by mixing with additional penetration enhancers, such asunconjugated forms of the lipid moieties described above, includingfatty acids and their derivatives. Examples include oleic acid, lauricacid, capric acid, myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, recinleate, monoolein(a.k.a. 1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arichidonicacid, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one,acylcarnitines, acylcholines, mono- and di-glycerides andphysiologically acceptable salts thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.).

Complex formulations comprising one or more penetration enhancing agentscan be used. For example, bile salts can be used in combination withfatty acids to make complex formulations. Exemplary combinations includechenodeoxycholic acid (CDCA), generally used at concentrations of about0.5 to 2%, combined with sodium caprate or sodium laurate, generallyused at concentrations of about 0.5 to 5%.

Pharmaceutical compositions and/or formulations comprising theriboamidates can also include chelating agents, surfactants andnon-surfactants. Chelating agents include, but are not limited to,disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines). Surfactants include, for example, sodiumlauryl sulfate, polyoxyethylene-9-lauryl ether andpolyoxyethylene-20-cetyl ether; and perfluorochemical emulsions, such asFC-43. Non-surfactants include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives, and non-steroidalanti-inflammatory agents, such as diclofenac sodium, indomethacin, andphenylbutazone.

Thus, in another aspect of the invention, there is provided a method offormulating a pharmaceutical composition, the method comprisingproviding a compound as described herein, and combining the compoundwith a pharmaceutically acceptable excipient. The compound can beprovided at pharmaceutical purity, as defined below. The method canfurther comprise adding to the compound, either before or after theaddition of the excipient, a penetration enhancing agent.

The pharmaceutical composition will typically comply with pharmaceuticalpurity standards. For use as an active ingredient in a pharmaceuticalpreparation, a compound such as those described in the present inventionis generally purified away from other reactive or potentiallyimmunogenic components present in the mixture in which they areprepared. Typically, to achieve pharmaceutical purity where a nucleicacid-based compound is the active ingredient, the active ingredient isprovided in at least about 50% homogeneity, for example 60%, 70%, 80% or90% homogeneity, as determined by functional assay, chromatography, orgel electrophoresis. The active ingredient is then compounded into amedicament in accordance with generally accepted procedures for thepreparation of pharmaceutical preparations. Thus, in accordance with thepresent invention, providing the compounds at pharmaceutical purityrequires that the compound be provided at at least about 50%homogeneity, for example at least 80% or 90% homogeneity.

The pharmaceutical composition will also typically be aliquoted andpackaged in either single dose or multi-dose units. The dosagerequirements for treatment with the riboamidate compound vary with theparticular compositions employed, the route of administration, theseverity of the symptoms presented, the form of the compound and theparticular subject being treated.

Pharmaceutical compositions as disclosed herein can be administered to asubject in a formulation and in an amount effective to achieve aclinically desirable result. The amount of compound per dose and thenumber of doses required to achieve such effects will vary depending onmany factors including the disease indication, characteristics of thepatient being treated and the mode of administration. Typically, theformulation and route of administration will provide a localconcentration at the disease site of between 1 μM and 1 nM of thecompound.

In general, the compounds are administered at a concentration thataffords effective results without causing any harmful or deleteriousside effects. Such a concentration can be achieved by administration ofeither a single unit dose, or by the administration of the dose dividedinto convenient subunits at suitable intervals throughout the day.

What is claimed is:
 1. An isolated small interfering RNA (siRNA)comprising an oligonucleotide of 19 to 25 nucleotides in length that iscomplementary to a target nucleic acid sequence, wherein: all of thenucleosides of the oligonucleotide are of the formula:

wherein each R₁ is independently chosen from fluorine and OR₂, R₂ ischosen from hydrogen and lower alkyl, and B is chosen from purines,pyrimidines, and analogs thereof; and at least one internucleosidelinkage in the oligonucleotide is a ribo-N3′→P5′ phosphoramidate (NP)linkage; and wherein the small interfering RNA is selected from asingle-stranded antisense form, or a double-stranded form comprisingboth sense and antisense strands wherein at least one strand comprisesthe oligonucleotide.
 2. The small interfering RNA according to claim 1,wherein all of the internucleoside linkages in the oligonucleotide arechosen from ribo-N3′→P5′ phosphoramidate (NP) and ribothiophosphoramidate (NPS) linkages.
 3. The small interfering RNAaccording to claim 1, wherein the RNA further comprises at least onecovalently conjugated lipid moiety.
 4. The small interfering RNAaccording to claim 3, wherein the at least one lipid moiety iscovalently conjugated to the 5′ or 3′ terminus of the RNA, and the atleast one lipid moiety is chosen from fatty acids, sterols andhydrocarbons.
 5. The small interfering RNA according to claim 3,comprising the structure:O-(x-L)n wherein O is the oligonucleotide; L is a lipid moiety; x is anoptional linker; and n is an integer ranging from 1 to 5, wherein ifn>1, each additional (x-L) component may be, independently, the same ordifferent.
 6. The small interfering RNA according to claim 5, wherein Lis a lipid chosen from substituted and unsubstituted fatty acids andsterols; or wherein L is chosen from substituted and unsubstitutedhydrocarbons.
 7. The small interfering RNA according to claim 6, whereinL is chosen from fatty acids substituted with at least one fluorine; orwherein L is chosen from hydrocarbons substituted with at least onefluorine.
 8. The small interfering RNA according to claim 5, wherein atleast 60% of the nucleobases in the oligonucleotide are ribonucleobases.9. A method for effecting the post-transcriptional silencing of at leastone gene, comprising administering to a mammal in need of suchpost-transcriptional silencing at least one small interfering RNAaccording to claim
 1. 10. The method of claim 9, wherein the smallinterfering RNA further comprises at least one covalently conjugatedlipid moiety.
 11. The method of claim 9, wherein the at least one geneencodes at least one mRNA chosen from cellular mRNAs and viral mRNAs; orwherein the at least one gene is an oncogene; or wherein the at leastone gene is a viral gene.
 12. A method for effecting thepost-transcriptional silencing of at least one gene, comprisingadministering to a mammal in need of such post-transcriptional silencingat least one small interfering RNA according to claim
 5. 13. The methodaccording to claim 12, wherein the at least one gene encodes at leastone mRNA chosen from cellular mRNAs and viral mRNAs; or wherein the atleast one gene is an oncogene; or wherein the at least one gene is aviral gene.
 14. The small interfering RNA according to claim 1, whereinsaid small interfering RNA is in single-stranded form, and is effectiveto inhibit the expression of an endogenous mammalian target RNAsequence.
 15. The single-stranded small interfering RNA according toclaim 14, wherein the small interfering RNA further comprises at leastone covalently conjugated lipid moiety.
 16. The single-stranded smallinterfering RNA according to claim 14, wherein the target RNA sequenceis encoded by a human gene.
 17. The small interfering RNA according toclaim 1, wherein said small interfering RNA is in double-stranded form,and is effective to inhibit the expression of an endogenous mammaliantarget RNA sequence.
 18. The double-stranded small interfering RNAaccording to claim 17, wherein the target RNA sequence is encoded by ahuman gene.
 19. The double-stranded small interfering RNA according toclaim 17, wherein the RNA further comprises at least one covalentlyconjugated lipid moiety.
 20. A small interfering RNA as recited in claim1 wherein said target nucleic acid sequence is a human immunodeficiencyvirus (HIV) gene, such that said siRNA modulates expression of said HIVgene.
 21. The small interfering RNA according to claim 20, wherein thesmall interfering RNA further comprises at least one covalentlyconjugated lipid moiety.
 22. A small interfering RNA as recited in claim1 wherein said target nucleic acid sequence is a beta site APP-cleavingenzyme (BACE) gene, such that said siRNA modulates expression of saidBACE gene.
 23. The small interfering RNA according to claim 22, whereinthe small interfering RNA further comprises at least one covalentlyconjugated lipid moiety.
 24. A small interfering RNA as recited in claim1 wherein said target nucleic acid sequence is an EGFR gene, such thatsaid siRNA modulates expression of said EGFR gene.
 25. The smallinterfering RNA according to claim 24, wherein the small interfering RNAfurther comprises at least one covalently conjugated lipid moiety.
 26. Asmall interfering RNA as recited in claim 1 wherein said target nucleicacid sequence encodes K-Ras, such that said siRNA modulates expressionof said K-Ras.
 27. The small interfering RNA according to claim 26,wherein the small interfering RNA further comprises at least onecovalently conjugated lipid moiety.
 28. A small interfering RNA asrecited in claim 1 wherein said target nucleic acid sequence is aprostaglandin D2 receptor (PTGDR) gene, such that said siRNA modulatesexpression of said PTGDR gene.
 29. The small interfering RNA accordingto claim 28, wherein the small interfering RNA further comprises atleast one covalently conjugated lipid moiety.
 30. A small interferingRNA as recited in claim 1 wherein said target nucleic acid sequence isan ADORA1 gene, such that said siRNA modulates expression of said ADORA1gene.
 31. The small interfering RNA according to claim 30, wherein thesmall interfering RNA further comprises at least one covalentlyconjugated lipid moiety.
 32. The small interfering RNA according toclaim 1, wherein at least one nucleoside comprises the formula

wherein R₁ is selected from fluorine and OR₂ wherein R₂ is methyl. 33.The small interfering RNA according to claim 2, wherein all of theinternucleoside linkages are ribo-N3′→P5′ phosphoramidate (NP) linkages.34. The small interfering RNA according to claim 33, wherein at leastone nucleoside comprises the formula

wherein R₁ is selected from fluorine and OR₂ wherein R₂ is methyl. 35.The small interfering RNA according to claim 1, wherein at least one ofthe internucleoside linkages is a ribo-N3′→P5′ thiophosphoramidate (NPS)linkage.
 36. The small interfering RNA according to claim 34, wherein atleast one nucleoside comprises the formula

wherein R₁ is selected from fluorine and OR₂ wherein R₂ is methyl. 37.The small interfering RNA according to claim 35, wherein all of theinternucleoside linkages in the oligonucleotide are chosen fromribo-N3′→P5′ phosphoramidate (NP) and ribo N3′→P5′ thiophosphoramidate(NPS) linkages.
 38. The small interfering RNA according to claim 37,wherein at least one nucleoside comprises the formula

wherein R₁ is selected from fluorine and OR₂ wherein R₂ is methyl.